The present invention belongs to the field of modulation of inflammatory processes, in particular of inflammatory airway diseases, in which macrophages play an important role. The inflammatory processes can be modulated according to the invention by influencing the function of receptors on macrophages, which receptors are identified to be involved in the inflammatory process.
Inflammatory processes involve a cascade of reactions. A wide variety of factors are involved in inflammatory processes leaving a single treatment to avoid said factors unsuccessful. This is in particular true for inflammatory processes of the airways, like the chronic inflammatory airway diseases.
Chronic inflammatory airway diseases include Chronic Bronchitis and Chronic Obstructive Pulmonary Disease (COPD). For example, COPD is a complex disease encompassing symptoms of several disorders: chronic bronchitis which is characterized by cough and mucus hypersecretion, small airway disease, including inflammation and peribronchial fibrosis, and emphysema. COPD is characterized by an accelerated and irreversible decline of lung function. The major risk factor for developing COPD is continuous cigarette smoking. Since only about 20% of all smokers are inflicted with COPD, a genetic predisposition is also likely to contribute to the disease.
The initial events in the early onset of COPD are inflammatory, affecting small and large airways. An irritation caused by cigarette smoking attracts macrophages and neutrophils the number of which is increased in the sputum of smokers. Perpetual smoking leads to an ongoing inflammatory response in the lung by releasing mediators from macrophages, neutrophils and epithelial cells that recruit inflammatory cells to sites of the injury. So far there is no therapy available to reverse the course of COPD. Smoking cessation may reduce the decline of lung function. Only a few drugs provide some relief for patients. Longlasting xcex22-agonists and anticholinergics are applied to achieve a transient bronchodilatation. A variety of antagonists for inflammatory events are under investigation like, LTB4-, IL-8-, TNFxcex1-inhibitors.
Chronic inflammatory airway diseases can be attributed to activated inflammatory immune cells, e.g. macrophages. There is a need for modulating the function of macrophages in order to eliminate a basis for inflammatory processes.
The present invention relates to substances which modulate receptors involved in inflammatory processes and whose modulated functions positively influence inflammatory diseases.
In the present invention it was found that macrophages involved in an inflammatory process, preferably in a chronic inflammatory airway disease, more preferably in chronic bronchitis or COPD, show a pattern of differentially expressed nucleic acid sequence and protein expression which differs from the pattern of gene expression of macrophages from healthy donors or donors in an irritated status, which latter do contain macrophages in an activated status. Therefore, macrophages show different activation levels under different inflammatory conditions, and it is shown in the present invention that macrophages in a hyperactive status are involved in an inflammatory process, preferably in a chronic inflammatory airway disease, more preferably in chronic bronchitis or COPD. The present invention provides for the inhibition of the hyperactivation or the reduction of the hyperactive status of a macrophage by allowing the identification of substances which modulate receptors involved in the hyperactivation or maintaining the hyperactive status.
The invention is based on the identification of a differentially expressed nucleic acid sequence or protein which is involved in causing the induction and/or maintenance of the hyperactive status of macrophages involved in an inflammatory process, preferably in a chronic inflammatory airway disease, more preferably in chronic bronchitis or COPD. Such differentially expressed nucleic acid sequence or protein is in the following named differentially expressed nucleic acid sequence or protein of the invention respectively. In particular, the present invention teaches a link between phenotypic changes in macrophages due to differentially expressed nucleic acid sequence and protein expression pattern and involvement of macrophages in inflammatory processes and, thus, provides a basis for a variety of applications. For example, the present invention provides a method and a test system for determining the expression level of a macrophage protein or differentially expressed nucleic acid sequence of the invention and thereby provides e.g. for methods for diagnosis or monitoring of inflammatory processes with involvement of hyperactivated macrophages in mammalian, preferably human beings, especially such beings suffering from an inflammatory process, preferably in a chronic inflammatory airway disease, more preferably in chronic bronchitis or COPD. The invention also relates to a method for identifying a substance by means of a differentially expressed nucleic acid sequence or protein of the invention processes, which substance modulates, i.e. acts as an inhibitor or activator on the said differentially expressed nucleic acid sequence or protein of the invention and thereby positively influences chronic inflammatory processes by inhibition of the hyperactivation or reduction of the hyperactive status of macrophages, and thereby allows treatment of mammals, preferably human beings, suffering from a said disease. The invention also relates to a method for selectively modulating such a differentially expressed nucleic acid sequence or protein of the invention in a macrophage comprising administering a substance determined to be a modulator of said protein or differentially expressed nucleic acid sequence. The present invention includes the use of said substances for treating beings in need of a treatment of an inflammatory process, preferably a chronic inflammatory airway disease, more preferably chronic bronchitis or COPD.
For the present invention in a first step differentially expressed nucleic acid sequences and proteins are identified which have a different expression pattern in a hyperactivated macrophage compared to a macrophage which is not hyperactivated. For the sake of conciseness this description deals particularly with investigation of macrophages involved in COPD, however, equivalent results may be observed with samples from patients suffering from other chronic inflammatory airway diseases, e.g. chronic bronchitis. The investigation of the different expression pattern leads to the identification of a series of differentially expressed nucleic acid sequences in macrophages, differentially expressed in dependency on the activation status of a macrophage involved in an inflammatory process, as exemplified in the Examples hereinbelow.
Briefly, such a differentially expressed nucleic acid sequence is identified by comparative expression profiling experiments using a cell or cellular extract from a hyperactivated macrophage, i.e. for example from the site of inflammation in a COPD and from the corresponding site of control being not suffering from said disease, however, suffering from an irritated condition like cigarette smoke exposure.
A differentially expressed nucleic acid sequence or protein of the invention can easily be detected by such a method because amongst the differentially expressed macrophage genes a class of differentially expressed nucleic acid sequences can be identified which encodes a class of macrophage surface receptors which is characterized in that it is expressed at a lower or higher level than the control level in a macrophage which is not hyperactivated. Such a macrophage surface receptor of the invention is hereinafter named ILM receptor. However, the invention does not only concern a naturally occurring ILM receptor, but also includes within the meaning of ILM receptor a receptor which is functionally equivalent to, i.e. which shares the binding capacities and the cellular function with an ILM receptor.
An example for an ILM receptor according to the present invention is a FPRL-1 receptor type receptor including FPRL-1 receptor (SEQ ID NO:2). The term xe2x80x9creceptor type receptorxe2x80x9d used in context with the present invention, e.g. FPRL-1 receptor type receptor, is a receptor which is xe2x80x9cfunctionally equivalentxe2x80x9d to, i.e. which shares the binding capacities and the cellular function with, the respective receptor, e.g. FPRL-1 receptor of SEQ. ID NO:2; the term also encompasses variants, mutants or fragments of a naturally occuring receptor, e.g. FPRL-1 receptor. or naturally occuring receptor type receptor, e.g. FPRL-1 receptor type receptor, which variants, mutants or fragments are functionally equivalent to the receptor, e.g. FPRL-1 receptor.
Further examples for ILM receptors are HM74 receptor type receptor including HM74 receptor (SEQ ID NO:21); AICL receptor type receptor including AICL receptor (SEQ ID NO:6); ILT1 receptor type receptor including ILT1 receptor (SEQ ID NO:12); SHPS-1 receptor type receptor including SHPS-1 receptor (SEQ ID NO:4); KDEL receptor 1 type receptor including KDEL receptor 1 (SEQ ID NO:8); and CSF-1 receptor type receptor including CSF-1 receptor (SEQ ID NO:10). Preferred is the respective receptor shown in the sequence listing or a variant, mutant or fragment thereof having the same function, even more preferred is the respective receptor shown in the sequence listing under SEQ ID NOs:21, 6, 12, 4, 8, 10. In even more preferred embodiments the receptors are encoded by the nucleic acid sequences having the SEQ ID NOs:20, 5, 11, 3, 7 or 9, respectively.
A preferred embodiment of an ILM receptor in context with the present invention is a FPRL-1 receptor type receptor. The term FPRL-1 receptor type receptor accordingly also encompasses variants, mutants or fragments, of naturally occuring FPRL-1 receptor or FPRL-1 receptor type receptors, which variants, mutants or fragments are functionally equivalent to the FPRL-1 receptor. An even more preferred embodiment in context with the description of the embodiments of the present invention is the FPRL-1 receptor of SEQ ID NO:2 or a variant, mutant or fragment thereof having the same function, even more preferred is the FPRL-1 receptor of SEQ ID NO:2. In a most preferred embodiment, the FPRL-1 receptor is encoded by the nucleic acid sequence shown in SEQ ID NO:1.
According to the present invention, the function of an ILM receptor expressed at a lower level than the control level is preferably activated in order to inhibit hyperactivation or reduce a hyperactivated status of a macrophage, whereby the function of an ILM receptor which is expressed at a higher level than the control level is preferably inhibited in order to inhibit hyperactivation or reduce a hyperactivated status of a macrophage. A function of a receptor in context with the present invention is any function of a receptor of the invention which is capable of influencing the inflammatory processes. For example, a receptor of the invention mediates inflammation in that it is activated by a ligand (any substance which has the capacity to bind to said receptor to at least one of its domains exposed on the cell surface) and leads to an intracellular signal involved in inflammatory processes.
In one embodiment the present invention concerns a method for determining a substance to be an activator or inhibitor of an ILM receptor characterized in that the receptor is deregulated preferably overexpressed or downregulated in a macrophage involved in a chronic inflammatory airway disease and which receptor plays a role in mediating inflammation. A method according to the invention comprises the application of a substance of interest to a test system which generates a measurable read-out upon modulation of the ILM receptor or of an ILM receptor function. A test system useful for performing such method of the invention comprises a cell or a cell-free system. For example, in one embodiment according to the invention the system is designed in order to allow the testing of substances acting on the expression level of the differentially expressed nucleic acid sequence, in another embodiment the system allows the testing of substances directly interacting with the receptor or interfering with the binding of the receptor with a natural or an artificial but appropriate ligand. The latter system comprises a receptor of the invention in a way that a substance which should be tested can physically contact said receptor and which direct interaction leads to a measurable read-out indicative for the change of receptor function.
A method according to the invention comprising a cellular system can be, for example, a method in which a MonoMac6 or a THP-1 cell is used wherein said cell is stimulated with phorbol 12-myristate 13-acetate and with a substance selected from a group consisting of LPS and smoke.
The present invention also provides a test system for determining whether a substance is an activator or an inhibitor according to the invention of an ILM receptor function according to the invention, characterized in that the receptor is involved in a chronic inflammatory airway disease and which receptor plays a role in mediating inflammation, comprising at least an ILM receptor or an expression vector capable of expressing an ILM receptor in a cell or a host cell transformed with an expression vector capable of expressing an ILM receptor.
For performing a method for determining whether a substance is an activator or an inhibitor of receptor function of the present invention cells as well as cell-free systems can be used. Test systems for performing the method can be, for example, designed and built up by using elements and methods well known in the art. For example, cell-free systems may include, for example, cellular compartments or vesicles comprising a receptor of the invention. Suitable cellular systems include, for example, a suitable prokaryotic cell or eukaryotic cell, i.e. comprising a respective receptor of the invention. A cell suitable for performing a said method of the invention may be obtained by recombinant techniques, i.e. after transformation or transfection with a vector suitable for expression of the desired receptor of the invention, or may be a cell line or a cell isolated from a natural source expressing the desired receptor of the invention. A test system according to the invention comprising a cellular system can also be, for example, a test system in which a MonoMac6 or a THP-1 cell is used wherein said cell is stimulated with phorbol 12-myristate 13-acetate and with a substance selected from a group consisting of LPS and smoke. A test system according to the invention may include a natural or artificial ligand of the receptor if desirable or necessary for testing whether a substance of interest is an inhibitor or activator of a receptor of the invention. Test systems of the invention may be availbale as kits.
A test method according to the invention comprises measuring a read-out, i.e. a phenotypic change in the test system, for example, if a cellular system is used a phenotypic change of the cell. Such change may be a change in a naturally occurring or artificial response of the cell to receptor activation or inhibition, e.g. as detailed in the Examples hereinbelow.
A test method according to the invention can on the one hand be useful for high throughput testing suitable for determining whether a substance is an inhibitor or activator of the invention, but also e.g. for secondary testing or validation of a hit or lead substance identified in high throughput testing.
The present invention also concerns a substance identified in a method according to the invention to be an inhibitor or activator of a receptor of the invention. A substance of the present invention is any compound which is capable of activating or preferably inhibiting a function of a receptor according the invention. An example of a way to activate or inhibit a function of a receptor is by influencing the expression level of said receptor. Another example of a way to activate or inhibit a function of a receptor is to apply a substance which directly binds the receptor, thereby activating or blocking functional domains of said receptor, which can be done reversibly or irreversibly, depending on the nature of the substance applied.
Accordingly, a substance useful for activating or inhibiting receptor function includes substances acting on the expression of a differentially expressed nucleic acid sequence, but also acting on the receptor itself. Therefore, according to the invention the meaning of the term a xe2x80x9csubstance of the inventionxe2x80x9d includes but is not limited to nucleic acid sequences coding for the gene of a receptor of the invention or a fragment or variant thereof and being capable of influencing the gene expression level, e.g. nucleic acid molecules suitable as antisense nucleic acid, ribozyme, or for triple helix formation. Another substance of the invention is e.g. an antibody or an organic or inorganic compound directly binding to or interfering with the binding of an appropriate ligand with a receptor of the invention and thereby affecting its function.
In a further aspect, the present invention relates to a method for determining an expression level of an ILM receptor differentially expressed nucleic acid sequence or protein according to the invention comprising determining the level of said ILM receptor in a macrophage according to the invention. Such a method can be used, for example, for testing whether a substance is capable of influencing differentially expressed nucleic acid sequence expression levels in a method outlined above for determining whether a substance is an activator or inhibitor. A method for determining an expression level of an ILM receptor differentially expressed nucleic acid sequence or protein can, however, also be used for testing the activation status of a macrophage, e.g. for diagnostic purposes or for investigation of the success of treatment of a disease which is caused by the hyperactivated macrophage.
Accordingly, the invention also relates to a method for diagnosis of a chronic inflammatory disease or monitoring of such disease, e.g. monitoring success in treating beings in need of treatment of such disease, comprising determining the level of the receptor expressed in a macrophage according to the invention. Said macrophage is preferably a mammalian, more preferably a human cell. Accordingly, macrophages of the present invention are preferably obtainable from the site of inflammation in a mammal and more preferably from a site of inflammation in a human being.
A method for determining expression levels of a receptor according to the invention can, depending on the purpose of determining the expression level, be performed by known procedures such as measuring the concentration of respective RNA transcripts via hybridization techniques or via reporter gene driven assays such as luciferase assays or by measuring the protein concentration of said receptor using respective antibodies to verify the identity of said protein.
The present invention relates to the use of a substance according to the invention for the treatment of a chronic inflammatory airways disease according to the invention. Another embodiment of the present invention relates to a pharmaceutical composition comprising at least one of the substances according to the invention determined to be an activator or an inhibitor using the method for determining whether the substance is an activator or an inhibitor according to the invention characterized in that the respective receptor according to the invention is overexpressed in a macrophage according to the invention involved in a chronic inflammatory airway disease according to the invention. The composition may be manufactured in a manner that is itself known, e.g. by means of conventional mixing, dissolving, granulating, dragee-making, levigating, powdering, emulsifying, encapsulating, entrapping or lyophilizing processes.
In order to use substances activating or inhibiting according to the invention as drugs for treatment of chronic inflammatory airway diseases, the substances can be tested in animal models for example an animal suffering from an inflammatory airway disorder or a transgenic animal expressing a receptor according to the invention.
Toxicity and therapeutic efficacy of a substance according to the invention can be determined by standard pharmaceutical procedures, which include conducting cell culture and animal experiments to determine the IC50, LD50 and ED50. The data obtained are used for determining the animal or more preferred the human dose range, which will also depend on the dosage form (tablets, capsules, aerosol sprays ampules, etc.) and the administration route (for example transdermal, oral, buccal, nasal, enteral, parenteral, inhalative, intratracheal, or rectal).
A pharmaceutical composition containing a least one substance according to the invention as an active ingredient can be formulated in conventional manner. Methods for making such formulations can be found in manuals, e.g. xe2x80x9cRemington Pharmaceutical Sciencexe2x80x9d. Examples for ingredients that are useful for formulating at least one substance according to the present invention are also found in WO 99/18193, which is hereby incorporated by reference.
In a further aspect the invention teaches a method for treating a chronic inflammatory airway disease according to the invention which method comprises administering to a being preferably to a human being in need of such treatment a suitable amount of a pharmaceutical composition comprising at least one substance determined to be an activator or inhibitor according to a method for determining whether a substance is an activator or an inhibitor according to the invention of an ILM receptor according to the invention characterized in that the receptor is overexpressed in a macrophage according to the invention and plays a role in mediating inflammation involved in a chronic inflammatory airway disease according to the invention.
In another embodiment the invention relates to a method for selectively modulating ILM receptor concentration in a macrophage, comprising administering a substance determined to be an activator or inhibitor of a receptor according to the invention.
Included herein are exemplified embodiments, which are intended as illustrations of single aspects of the invention. Indeed, various modifications of the invention in addition to those herein will become apparent to those skilled in the art from the foregoing description and drawings. Such modifications are intended to fall within the scope of the present invention.
All publications and patent applications cited herein are incorporated by reference in their entireties.